Over 2 million long bone fractures are treated in the United States every year. Although most bone fractures heal spontaneously there is no gold standard for promoting bone regeneration in those settings in which either fractures do not heal or there is a critical sized segmental bone defect due to trauma or infection, devastating medical problems leading to significant disability. The recent development of custom printed biomaterial scaffolds that can fit and fill large bone defects may provide a novel solution and coating these scaffolds with agents designed to promote more rapid and complete bone healing may increase the efficacy of prosthetic scaffolds in healing segmental bone defects. Although currently used to promote bone generation, growth factors such as rh-BMP2 (BMP2) are of questionable efficacy and present significant safety issues. We have recently reported that adenosine A2A receptor (A2AR) stimulation increases osteoblast number and regulates osteoblast function in a murine model of inflammatory osteolysis and that A2AR stimulation diminishes osteoclast differentiation by inhibiting NFB activation and nuclear translocation. Moreover, A2ARs stimulate angiogenesis and vasculogenesis in vitro and in vivo. Thus, we propose to test the hypothesis that 3- dimensional printed scaffolds coated with an agent, dipyridamole, that increases local adenosine levels and indirectly stimulates A2ARs can further promote bone regeneration at critical sized segmental bone defects and to determine the cellular and molecular mechanisms for this phenomenon. We therefore propose the following aims: I. Development of coated bioactive ceramic scaffolds to treat critical segmental bone defects. We will determine whether implanting 3-dimensionally printed calcium triphosphate/hydroxyapatite scaffolds coated with dipyridamole, an agent which blocks cellular adenosine uptake and increases adenosine concentration in extracellular fluids, promotes bone regeneration in a rabbit radius model of segmental bone defect. We will further maximize scaffold design and dipyridamole dosing in vitro and in a murine calvaria model of bone regeneration. II. Determination of the cellular mechanism by which A2AR stimulation promotes bone regeneration. Using global and cell-selective knockouts of A2AR we will determine the cellular basis for A2AR-mediated bone regeneration in the murine calvaria model. III. Examination of the molecular mechanisms by which A2AR stimulation promotes bone regeneration in osteoblasts. We will test the hypothesis that A2AR signaling interacts with critical intracellular signaling cascades to promote bone regeneration using pharmacologic inhibitors of signaling pathways and by targeted knockdown of critical signaling molecules in primary cells and cell lines. The goals of this highly translational project are to establish the molecular and cellular basis for targeting A2ARs to stimulate bone regeneration and to rapidly translate these findings to the clinic.